Differentiation of live and dead cells is an important challenge in microbial diagnostics. Metabolic and reproductive activity, and, in the case of pathogenic microorganisms, the potential health risk are limited to the live portion of a mixed microbial population. Four physiological states are distinguished in flow cytometry using fluorescent stains: reproductively viable, metabolically active, intact and permeabilized cells. Depending on the conditions, all stages except the permeabilized cells can have the potential of recovery upon resuscitation and thus have to be considered potentially live8. Due to the relatively long persistence of DNA after cell death in the range between days to 3 weeks4,6, DNA-based diagnostics tend to overestimate the number of live cells. DNA extracted from a sample can originate from cells in any of the four mentioned physiological states including the dead permeabilized cells. Detection of the latter, however, is not desired.
The most important criterion for distinguishing between viable and irreversibly damaged cells is membrane integrity. Sorting out noise derived from membrane-compromised cells helps to assign metabolic activities and health risks to the intact and viable portion of bacterial communities. Live cells with intact membranes are distinguished by their ability to exclude DNA-binding dyes that easily penetrate dead or membrane-compromised cells. This principle is routinely applied for microscopic live-dead discrimination and increasingly in flow-cytometry. The most common membrane-impermeant dye is propidium iodide.
In the last few years, EMA-PCR was reported to be an easy-to-use alternative to microscopic or flow-cytometric distinction between live and dead cells11,13,14. This diagnostic DNA-based method combines the use of a live-dead discriminating dye with the speed and sensitivity of real-time PCR. Ethidium monoazide (EMA) is a DNA-intercalating dye with the azide group allowing covalent binding of the chemical to DNA upon exposure to bright visible light (maximum absorbance at 460 nm). Cells are exposed to EMA for 5 minutes allowing the dye to penetrate dead cells with compromised cell walls/membranes and to bind to their DNA. Photolysis of EMA using bright visible light produces a nitrene that can form a covalent link to DNA and other molecules1,2. Photo-induced cross-linking was reported to inhibit PCR amplification of DNA from dead cells. In a recent publication it could be shown that EMA-crosslinking to DNA actually rendered the DNA insoluble and led to its loss together with cells debris during genomic DNA extraction10. The unbound EMA, which remains free in solution, is simultaneously inactivated by reacting with water molecules2. The resulting hydroxylamine is no longer capable of covalently binding to DNA5. DNA from viable cells, protected from reactive EMA before light-exposure by an intact cell membrane/cell wall, is therefore not affected by the inactivated EMA after cell lysis. EMA treatment of bacterial cultures comprised of a mixture of viable and dead cells thus leads to selective removal of DNA from dead cells. The species tested were E. coli 0157:H711, Salmonella typhimurium11, Listeria monocytogenes11,13,14 and Campylobacter jejuni13. These studies did not examine, however, the selective loss of DNA from dead cells.
Though this technique is promising, the use of EMA prior DNA extraction was found to suffer from a major drawback. In the case of E. coli 0157:H7, though the entire genomic DNA from dead cells was removed, the treatment also resulted in loss of approximately 60% of the genomic DNA of viable cells harvested in log phase10. It was observed in this study that EMA also readily penetrates viable cells of other bacterial species resulting in partial DNA loss. The lack of selectivity and of overall applicability led to testing a newly developed alternative chemical: Propidium monoazide (PMA). PMA is identical to PI except that the additional presence of an azide group allows crosslinkage to DNA upon light-exposure. As PI is highly membrane impermeant and generally excluded from viable cells, it has been extensively used to identify dead cells in mixed populations. Upon penetrating compromised cell membranes, PI binds to DNA by intercalating between the bases with little or no sequence preference and with a stoichiometry of one dye molecule per 4-5 base pairs of DNA17.
The higher charge of the PMA molecule (2 positive charges compared to only one in the case of EMA) and the fact that selective staining of nonviable cells with propidium iodide (PI) has been successfully performed on a wide variety of cell types, gave confidence that the use of PMA might mitigate the drawbacks observed with EMA.
The invention examined the suitability of PMA to selectively remove detection of genomic DNA of dead cells from bacterial cultures with defined portions of live and dead cells. Because this is a newly developed molecule, optimization of the methods was necessary. Photo exposure time for DNA binding and simultaneous inactivation of free unbound PMA was optimized using purified DNA. PMA concentration and incubation time were optimized with one gram-negative and one gram-positive organism before applying these parameters to the study of a broad-spectrum of different bacterial species.